Variations In Sarcomere Length Research Articles

The Skeletal Muscle Molecular Clock Regulates Titin Splicing and Protein Expression

The circadian factor BMAL1 is a fundamental transcriptional regulator of cell time keeping and expression of cell specific genes important for homeostasis. Our lab developed the inducible, skeletal muscle specific Bmal1 knockout (iMSBmal1-/-) mouse to determine the role of this protein in adult skeletal muscle. Skeletal muscles from these mice exhibit decreased specific tension and reduced unstimulated tension developed during a fatigability test. Since this change could be caused by changes in the elastic properties of the muscle, we investigated whether titin isoform expression changes in these mice. The tibialis anterior muscle of the iMSBmal1-/- mice exhibits a significant shift in expression from a short to long isoform of titin protein (38% long in iMSBmal1-/- vs. 19% in iMSBmal1+/+). RNA-Seq from these muscles indicates increased inclusion of titin exons 150-155 and 190-225, which are found within titin's PEVK region. Because this region is highly extensible and acts as a spring, we performed immunohistochemistry to test whether changes to this protein could have an effect on sarcomere length. An antibody to α-actinin was used to demarcate Z-lines in longitudinal sections of the tibialis anterior muscle and sarcomere length was measured as the distance between Z-lines. While average sarcomere length was not different between iMSBmal1+/+ and iMSBmal1-/- muscle, variation in sarcomere length increased following Bmal1 knockout. To test if increased variability of titin isoform expression similar to the iMSBmal1-/- mouse could lead to variable sarcomere lengths, we are currently transfecting C2C12 myoblasts with U7 snRNPs targeted to titin PEVK regions. Sarcomere length variability will be measured in these cells following 7 days of differentation. Current studies with this model are ongoing and will lead to increased knowledge of titin's role in regulating sarcomere length in a skeletal muscle at rest.

The Skeletal Muscle Molecular Clock Regulates Titin Isoform Expression

The circadian clock transcription factors, BMAL1 and CLOCK, are fundamental transcriptional regulators of cell time keeping and critical cell specific genes important for homeostasis. To determine the specific role of the molecular clock in adult skeletal muscle, our lab developed the inducible, skeletal muscle specific Bmal1 knockout (iMSBmal1-/-) mouse. Notably, skeletal muscles from these mice exhibit decreased specific tension and reduced unstimulated tension developed during a fatigability test. To begin to discern the molecular mechanisms that link the changes in the molecular clock with changes in muscle function, we tested whether iMSBmal1-/- muscle would also exhibit changes in sarcomeric protein expression. We determined that the tibialis anterior muscle of the iMSBmal1-/- mice exhibits a significant increase (38% in iMSBmal1-/- mice vs. 19% in iMSBmal1+/+ mice) in expression of a longer isoform of titin. It is established that titin, the giant filamentous protein that maintains sarcomeric structure, underlies passive tension, and contributes to active tension development. To determine if this increased heterogeneity of titin isoforms within the TA had an effect on sarcomere length, we performed immunohistochemistry. An antibody to α-actinin was used to demarcate Z-lines in longitudinal sections of the tibialis anterior muscle and sarcomere length was measured as the distance between Z-lines. While average sarcomere length was not different between iMSBmal1+/+ and iMSBmal1-/- muscle, variation in sarcomere length was increased following Bmal1 knockout in skeletal muscle. Further studies with our model are currently ongoing and will lead to increased knowledge of the importance of titin isoform maintenance in a variety of pathological conditions.

Stretching Skeletal Muscle: Chronic Muscle Lengthening through Sarcomerogenesis

Skeletal muscle responds to passive overstretch through sarcomerogenesis, the creation and serial deposition of new sarcomere units. Sarcomerogenesis is critical to muscle function: It gradually re-positions the muscle back into its optimal operating regime. Animal models of immobilization, limb lengthening, and tendon transfer have provided significant insight into muscle adaptation in vivo. Yet, to date, there is no mathematical model that allows us to predict how skeletal muscle adapts to mechanical stretch in silico. Here we propose a novel mechanistic model for chronic longitudinal muscle growth in response to passive mechanical stretch. We characterize growth through a single scalar-valued internal variable, the serial sarcomere number. Sarcomerogenesis, the evolution of this variable, is driven by the elastic mechanical stretch. To analyze realistic three-dimensional muscle geometries, we embed our model into a nonlinear finite element framework. In a chronic limb lengthening study with a muscle stretch of 1.14, the model predicts an acute sarcomere lengthening from 3.09m to 3.51m, and a chronic gradual return to the initial sarcomere length within two weeks. Compared to the experiment, the acute model error was 0.00% by design of the model; the chronic model error was 2.13%, which lies within the rage of the experimental standard deviation. Our model explains, from a mechanistic point of view, why gradual multi-step muscle lengthening is less invasive than single-step lengthening. It also explains regional variations in sarcomere length, shorter close to and longer away from the muscle-tendon interface. Once calibrated with a richer data set, our model may help surgeons to prevent muscle overstretch and make informed decisions about optimal stretch increments, stretch timing, and stretch amplitudes. We anticipate our study to open new avenues in orthopedic and reconstructive surgery and enhance treatment for patients with ill proportioned limbs, tendon lengthening, tendon transfer, tendon tear, and chronically retracted muscles.

Minimally invasive high-speed imaging of sarcomere contractile dynamics in mice and humans

Sarcomeres are the basic contractile units of striated muscle. Our knowledge about sarcomere dynamics has primarily come from in vitro studies of muscle fibres and analysis of optical diffraction patterns obtained from living muscles. Both approaches involve highly invasive procedures and neither allows examination of individual sarcomeres in live subjects. Here we report direct visualization of individual sarcomeres and their dynamical length variations using minimally invasive optical microendoscopy to observe second-harmonic frequencies of light generated in the muscle fibres of live mice and humans. Using microendoscopes as small as 350 microm in diameter, we imaged individual sarcomeres in both passive and activated muscle. Our measurements permit in vivo characterization of sarcomere length changes that occur with alterations in body posture and visualization of local variations in sarcomere length not apparent in aggregate length determinations. High-speed data acquisition enabled observation of sarcomere contractile dynamics with millisecond-scale resolution. These experiments point the way to in vivo imaging studies demonstrating how sarcomere performance varies with physical conditioning and physiological state, as well as imaging diagnostics revealing how neuromuscular diseases affect contractile dynamics.

Rat medial gastrocnemius muscles produce maximal power at a length lower than the isometric optimum length.

The interaction of relative muscle length and force-velocity characteristics was investigated in the fully activated rat medial gastrocnemius muscle in situ. Average maximal isometric force (as a percentage of the of the maximal isometric force at L(o,iso)) at relative lengths measured below isometric optimum (L(o,iso)) was 96% at L(o,iso)-2 mm, 88% at L(o,iso)-4 mm and 58% at L(o,iso)-6 mm. Force-velocity curves were obtained at the four relative muscle lengths. There were no significant differences in maximal shortening velocity (approximately 280 mm x s(-1)) between the different muscle lengths. The highest power output (P<0.05) was found at L(o,iso)-2 mm (mean+/-SEM 435+/-19 mW). Peak power values at L(o,iso) (390+/-10 mW) and L(o,iso)-4 mm (395+/-12 mW) were not significantly different, whereas peak power was lowest (P<0.05) at L(o,iso)-6 mm. There was a significant (P<0.01) shift of approximately 1.5 mm in optimum muscle length for force generation during shortening contractions compared with isometric contractions. Shortening velocity had only a minor influence on optimum muscle length for force generation. It is concluded that fully activated muscles produce their maximal power at a length lower than L(o,iso). The difference in optimum length between isometric and dynamic contractions may be related to length-dependent variations in sarcomere length in series during shortening.

CALCIUM CHELATORS AND SALT EXTRACTED MYOFIBRILLAR PROTEIN INJECTION INTO LEAN PRERIGOR BEEF MUSCLE: EFFECTS ON TENDERNESS INDICES

ABSTRACTPrerigor beef longissimus muscles were removed within 40 min postmortem and injected 10% (within 2 h postmortem) with either a chelator (alginate, 3%; haametaphosphate, 3%; EDTA, 0.13%; citrate, 0.13%), meat slurry (45%), or a myofibrillar meat extract (3.7% protein) to evaluate the effects of each injection on the physical and chemical properties compared to a control and a water only injected treatment. Gross muscle shortening was not inhibited The water only, meat extract and citrate treatments had shorter (P&lt;0.05) sarcomeres than the control. The meat shy, alginate, phosphate, and EDTA treatments produced sarcomere lengths equivalent to the control. The control had the lowest (P&lt;0. 05) pH and percentage moisture. However, there were no differences in percentage cooking losses. Although variations in sarcomere length were apparent, these treatments did not improve tenderness (P&gt;0.05) as measured by Warner‐Bratzler shear.

The effect of muscle length on intracellular calcium and force in single fibres from mouse skeletal muscle.

1. The effect of muscle length on the myoplasmic free Ca2+ concentration ([Ca2+]i) and tetanic force production of intact single muscle fibres was investigated in the mouse flexor brevis muscle. Muscle fibres were analysed at 100 microns intervals from 100 microns shorter than the optimum length (L(zero)) to 300 microns longer than L(zero). The fluorescent Ca2+ indicator indo-1 was used to measure [Ca2+]i. 2. Changes in muscle length did not significantly affect tetanic or resting [Ca2+]i. However, at lower stimulus frequencies (30-50 Hz) the force-length relation was shifted to longer muscle lengths. 3. Force-tetanic [Ca2+]i curves were constructed at each muscle length. A comparison of these curves revealed that the Ca2+ sensitivity was length dependent. Ca2+ sensitivity increased over the ascending limb and plateau phase of the force-length curve, but over the descending limb Ca2+ sensitivity reached a plateau and then started to decrease once more. 4. These results show that length-dependent changes in Ca2+ sensitivity of single muscle fibres cannot simply be related to variations in sarcomere length. It is proposed that other factors, such as cross-bridge attachment and/or developed force, have a role in determining the Ca2+ sensitivity at a particular muscle length.

Biophysical aspects of meat tenderness

Biophysical aspects of meat tenderness is reviewed, where the meat structural origin of variation in meat tenderness is tried to be elucidated. Processes, such as rigor development and ageing, known to influence the properties of the structural components, is covered, and variables that influence those processes, such as chilling, electrical stimulation and stress ante-mortem, are discussed. Meat tenderness can be evaluated both by sensory and instrumental methods. The relationships between mechanical and sensory assessments tend to be non-linear, which can be due to non-linearity in the sensory evaluation and that muscle fibre orientation is easier to control in instrumental than in sensory evaluation. Structural changes of the meat occuring during rigor development are both longitudinal and lateral contraction of the myofibrillar mass. Other structural events, based on the proteolytic action, are the loosing up of the myofibrils held together laterally, weakening of the myofibrillar length and myofibril fragmentation. Using instrumental recordings of meat toughness (Warner-Bratzler (W-B) peak force), it decreases significantly with degree of contraction, when raw, but the reverse is found, when meat is cooked above 60 °C. A structural explanation to this behaviour is suggested to be the following. When meat is raw the lateral contraction of the meat fiber increases with shorter sarcomeres, giving rise to a larger viscous component and hence a lower W-B peak force. On heating, however, with a larger extracellular space, when shortened, there is more room for the connective tissue to contract without being restricted by the myofibrillar mass. This in turn gives a higher number of fibers per unit cross-sectional area, hence a larger elastic modulus and a higher W-B peak force, when cooked. When chilling of muscle during rigor both warm- and cold-shortening occur. Minimal shortening region is for beef M. longissimus dorsi (LD) 10–15 °C and for M. semimembranosus (SM) 7–13 °C. For the SM muscle there is a high correlation between percentage shortening and ultimate tenderness both in the warm- and cold-shortening region. But for the LD muscle this is only the case in the cold-shortening region. This observation suggests that the LD muscle is a more enzymatically active muscle than SM. The influence of low-voltage electrical stimulation (ES) was followed in the cold-shortening region for muscles LD and SM. A significant effect on tenderness 15 days post-mortem was only observed for LD at 1 °C and 4 °C, but not for SM. It was suggested that enhanced proteolysis could be the reason for the improved tenderness on ES of LD, as colds-hortening was not prevented by ES. Long-term and short-term stress ante-mortem can give rise to DFD (dark, firm and dry)-and PSE (pale, soft and exudative)-meat, respectively. DFD-meat (pH u > 6.0 in LD) has relatively short sarcomere lengths, but still it is swollen laterally and has consequently a small extracellular space. Therefore DFD-meat usually is tender. PSE-meat has a large variation in sarcomere length. The long sarcomeres of PSE-meat is suggested to be caused by reduced shortening, due to the denaturation of the sarcoplasmic proteins during rigor. The short sarcomeres can be caused by a higher percentage of rigor development in the warm-shortening region and that the denaturation of the myosin heads cause both longitudinal and lateral contraction of the myofibrillar mass. There is also a large variation in tenderness of PSE-meat, but it has been found that this variation is positively correlated to the sarcomere length (r = 0.52**), as has been shown for the other variables that governs the rigor process.

Interval dependence of force and twitch duration in rat heart explained by Ca2+ pump inactivation in sarcoplasmic reticulum.

1. The influence of the interstimulus interval on twitch duration was analysed in isolated heart muscle of the rat. When the muscle was in the steady state at interstimulus intervals at 5 s a test interval was interposed and varied. Duration of twitch and action potential, sarcomere length and peak force of the test beats were measured. 2. Twitch force and duration increased when the test interval was increased from 0.4 to 10 s. This effect was abolished by inhibitors of sarcoplasmic reticulum function (ryanodine, caffeine, Sr2+). Hence, the interval dependence is controlled by the sarcoplasmic reticulum. 3. Post-extrasystolic potentiation, variation of [Ca2+]o and [Na+]o and blocking of iCa with nifedipine and Mn2+ led to large variations in force, reflecting variations in the amount of Ca2+ released from the sarcoplasmic reticulum. The effect on twitch duration was small, indicating that twitch duration was rather insensitive to the amount of released Ca2+, and not controlled by iCa and Na(+)-Ca2+ exchange. 4. Action potential duration was much shorter than twitch duration and, depending on the intervention, changes were in the same or in opposite direction. Hence, the action potential did not determine twitch duration. 5. Small variations in sarcomere length amongst test contractions were observed, but these variations could not account for the effects of the test interval. 6. It is proposed that the Ca2+ pump in the sarcoplasmic reticulum is activated during each contraction and inactivates slowly. Thus, after a short interval the pump is still activated and rapidly sequesters much of the released Ca2+ leading to a small twitch and rapid relaxation. This mechanism ensures proper relaxation and diastolic filling of the ventricle. The biochemical basis and implications of the hypothesis are discussed.

Light diffraction patterns and sarcomere length variation in striated muscle fibers of Limulus.

Light diffraction patterns produced by Limulus striated muscle fibers were examined. Segments of fibers were glycerinated, fixed or bathed in relaxing solution. Profiles of the intensity of a diffracted order vs. the angle of incidence of the laser beam often exhibited narrow peaks with the fiber at rest length. The incident angles at which the intensity of left and right orders is greatest are used to calculate the sarcomere length, supporting the notion that regions of the fiber are organised into Bragg reflecting planes. These profiles developed subpeaks and broadened upon stretch of the fiber. The broad angle scan profiles are suggested to result from a decrease in the regular packing of myofibrils as the fiber is lengthened. The angular width of the subpeaks is used to estimate the thickness of clusters of myofibrils. The variation in sarcomere length along the fiber, as determined by the 0th to 1st diffraction order spacing, was dependent upon the fiber preparation. Glycerinated fibers and those bathed in relaxing solution showed more variation than fixed fibers. The variation of sarcomere length is compared to the variation in thick filament lengths in Limulus reported by Dewey et al. (1982). These results are compared to those obtained from frog fiber segments.

Influence of the Physical State of Tissue during Rigor Mortis upon Protein Solubility and Associated Properties of Bovine Muscle

SUMMARY— Protein solubility and associated properties were studied in bovine sternomandibularis muscle allowed to pass into rigor in three physical states. Thirty min post‐mortem, samples were incubated at 7°C for 48 hr in one of the following conditions: minced through 1/8‐in. plate, free to shorten in a vertical position, stretched to 150% of equilibrium length. Stretched muscle exhibited greater protein solubility, higher pH values and longer sarcomeres than the remaining samples. For post‐rigor muscle, protein solubility may be related to sarcomere length and moisture press ratio. Variations in sarcomere length may be related to post‐mortem changes in pH. Possible relationships between the contractile state of proteins and the chemical, physical and quality characteristics of muscle are discussed.

Myocardial cell and sarcomere lengths in the normal dog heart.

The purpose of this study was to measure myocardial cell and sarcomere lengths at the bases and apices of the right (RV) and left ventricle (LV) of 4 normal canine hearts. The RV and LV were simultaneously fixed with glutaraldehyde at zero transmural pressure. Sections of trabeculae carneae were taken from the 1 cm cut at the bases (free wall) of the RV and LV. They were further fixed with osmium tetroxide, embedded in Epon 812, sectioned at ½ to 1 µ and stained with azure II and methylene blue. Measured from photomicrographs (X 1,000) taken with the phase microscope, mean cell length was 70.9 µ±1.49 (SEM); no statistical difference existed between the cell lengths at the bases and apices of the RV and LV. From adjacent tissue, the measurements of sarcomere lengths made from photomicrographs (phase microscope, oil immersion lens, enlarged to X 4,000) were not statistically different from those made with the electron microscope (P&gt;0.9). The sarcomere lengths at the LV base were the shortest-2.16 µ±0.002 (SEM), followed by the LV apex, 2.28 µ±0.005 (SEM); RV base, 2.41 µ±0.006 (SEM); and RV apex, 2.46 µ±0.003 (SEM). These variations in sarcomere length show the importance of specifying the site of sampling of myocardial tissue. The sarcomere lengths were related inversely to the thickness of the ventricular wall. This observation may be considered the structural reflection of the lesser distensibility of the left base as compared with the right apex, and may be simply the manifestation of the fact that the left base contains more muscle fibers.

Contractility and ultrastructure in glycerol--extracted muscle fibers. I. The relationship of contractility to sarcomere length.

This study was undertaken to determine whether glycerol-extracted rabbit psoas muscle fibers can develop tension and shorten after being stretched to such a length that the primary and secondary filaments no longer overlap. A method was devised to measure the initial sarcomere length and the ATP-induced isotonic shortening in prestretched isolated fibers subjected to a small preload (0.02 to 0.15 P(0)). At all degrees of stretch, the fiber was able to shorten (60 to 75 per cent): to a sarcomere length of 0.7 micro when the initial length was 3.7 micro or less, and to an increasing length of 0.9 to 1.8 micro with increasing initial sarcomere length (3.8 to 4.4 micro). At sarcomere lengths of 3.8 to 4.5 micro, overlap of filaments was lost, as verified by electron microscopy. The variation in sarcomere length within individual fibers has been assessed by both light and electron microscopic measurements. In fibers up to 10 mm in length the stretch was evenly distributed along the fiber, and with sarcomere spacings greater than 4 micro there was only a slight chance of finding sarcomeres with filament overlap. These observations are in apparent contradiction to the assumption that an overlap of A and I filaments is necessary for tension generation and shortening.